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Nine categories of pitfalls in initial genetic testing for rare disorders reporting inappropriate negative, inconclusive or positive results

Clinical Genetics and Therapeutics
  • Primary Categories:
    • Clinical Genetics
  • Secondary Categories:
    • Clinical Genetics
Introduction:
An increasing number of patients suspected with rare disorders visit or are referred to physicians bringing genetic testing results. Failure to identify causative variants or false negative results occurs, presenting significant challenges in interpreting these findings and using them to inform clinical decision-making.

Methods:
We categorized the factors contributing to inaccurate initial results in 21 patients. We performed exome/genome sequencing (ES/GS) data reanalysis, further GS, RNA sequencing (RNA-seq), and/or reverse deep phenotyping (RDP).

Results:
We corrected the diagnostic results for 21 patients with initial inappropriate negative, inconclusive or positive results. We identified nine categories of factors likely contributed to previously unidentified causative variants or false positive results.



1. Missed variant

1.1 Genotype

1.1.1 Non-coding variant

Patient 1 presented with Marfan-like features and carried a deep intronic VUS c.6872-1003C>T in FBN1. RNA-seq showed aberrant splicing, enabling the reclassification to likely pathogenic. Patients 2-4 had non-coding variants identified in BTK, SOST, and DMD, respectively.

1.1.2 Misalignment

Patient 5, clinically diagnosed with Allan-Herndon-Dudley syndrome, received four previous non-diagnostic results. We identified a hemizygous c.290_302delins variant in SLC16A2 inherited from his mother. The altered sequence showed 97% identity with 309_335inv, leading to misalignment.

1.1.3 Exon-level copy number variant (CNV)

Patient 6 had only one variant in ARSB reported from ES.  GS identified a 0.9 kb deletion encompassing exon 4 of ARSB. Patients 7 and 8, monozygotic twins, had a similar missed CNV in CCN6.

1.1.4 RNA-seq as prior diagnostic tool

Patient 9 presented with neuromuscular scoliosis. GS performed at an outside lab yielded negative results. We performed RNA-seq and revealed significantly decreased expression of SMN1. As we did not have his DNA data, we requested a reanalysis from the lab, with a focus on SMN1, which identified a homozygous deletion of exon 7. Similarly, RNA-seq led to the identification of disease-causing deletions in 1p34.3 and 16p11.2 for Patients 10 and 11, respectively.

1.1.5 Mosaicism

Patient 12 was referred with multiple systemic aneurysms. Previous GS reported multiple variants, including one in TGFBR1. Her mother was mosaic and “asymptomatic”. RDP revealed mild features indicating Loeys-Dietz syndrome (LDS) in both of them, leading to a diagnosis of LDS. Patient 14 presented with multiple giant nevi but received negative ES results. We ordered testing with a tissue sample and revealed a recurrent pathogenic c.181C>A variant in NRAS.



1.2 Overlooked Minor Phenotype

1.2.1 In family member

Patient 15 carried compound heterozygous VUSes in SLCO2A1, inherited from his “asymptomatic” parents. RDP identified clubbing toes in the father, a minor finding of Primary hypertrophic osteoarthropathy. Together with additional reported cases involving the same maternal variant, both variants were considered as probably disease-causing. Patient 16’ family received similar RDP and confirmed the pathogenicity of variants in COL7A1.

1.2.2 In proband

RDP in patient 17 identified mild vertebral dysplasia, facilitating the identification of a likely pathogenic variant in TRPV4. RDP with reanalysis for Patient 18 ruled out ACAN-associated Spondyloepimetaphyseal dysplasia and confirmed the pathogenicity of a variant in PLS3.



2. False positive

2.1 CNV calling

Initial GS for patients 19 and 20 reported disease-associated deletion in SCNN1B and TTN, respectively. Manual inspection of BAM files revealed false positives.

 

2.2 Overinterpretation

Patient 21 had hyperthyroidism and received a positive result showing a variant in TSHR. The variant had been demonstrated to be loss-of-function and led to hypothyroidism, not consistent with her presentations. Therefore, this variant was unlikely to be responsible for her hyperthyroidism.

 

Conclusion:
The nine categories of pitfalls raise warnings in the interpretation of genetic testing reports and offer insights for further investigation of genetic data in unsolved cases.

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